Kang-Jie Chen

Selective Recruitment of Regulatory T Cell through CCR6-CCL20 in Hepatocellular Carcinoma Fosters Tumor Progression and Predicts Poor Prognosis

Background Regulatory T cells (Tregs) are highly prevalent in tumor tissue and can suppress effective anti-tumor immune responses. However, the source of the increased tumor-infiltrating Tregs and their contribution to cancer progression remain poorly understood. Methodology/Principal Finding We here investigated the frequency, phenotype and trafficking property of Tregs and their prognostic value in patients with hepatocellular carcinoma (HCC). Our results showed that FoxP3+ Tregs highly aggregated and were in an activated phenotype (CD69+HLA-DRhigh) in the tumor site, where they can suppress the proliferation and INF-γ secretion of CD4+CD25− T cells. These tumor-infiltrating Tregs could be selectively recruited though CCR6-CCL20 axis as illustrated by (a) high expression of CCR6 on circulating Tregs and their selective migration to CCR6 ligand CCL20, and (b) correlation of distribution and expression between tumor-infiltrating Tregs and intratumoral CCL20. In addition, we found that the number of tumor-infiltrating Tregs was associated with cirrhosis background (P = 0.011) and tumor differentiation (P = 0.003), and was an independent prognostic factor for overall survival (HR = 2.408, P = 0.013) and disease-free survival (HR = 2.204, P = 0.041). The increased tumor-infiltrating Tregs predicted poorer prognosis in HCC patients. Conclusions The CCL20-CCR6 axis mediates the migration of circulating Tregs into tumor microenvironment, which in turn results in tumor progression and poor prognosis in HCC patients. Thus, blocking CCL20-CCR6 axis-mediated Treg migration may be a novel therapeutic target for HCC.

Effects of silicon on defense of wheat against oxidative stress under drought at different developmental stages

The effects of silicon application before sowing on the drought-induced oxidative stress and antioxidant defense in wheat (Triticum aestivum L.) were investigated. Drought stress was applied by withholding watering till sampling at booting or filling stage. Application of Si increased the water potential of drought-stressed plants at filling stage, whereas it did not at booting stage. The superoxide dismutase (SOD) activity was inhibited and peroxidase (POD) activity was enhanced by drought at booting stage, and no differences were observed due to the Si treatment. At filling stage, however, application of Si increased the SOD activity and decreased the POD activity of drought-stressed plants. The catalase (CAT) activity was slightly increased by drought only in the absence of Si and at booting stage. The activity of glutathione reductase (GR) was not greatly influenced. Application of Si did not change the contents of H2O2, total soluble protein and protein carbonyl of drought-stressed plants at booting stage, whereas at filling stage, it decreased the content of H2O2 and protein carbonyl and increased the content of total soluble protein. The content of thiobarbituric acid reactive substances (TBARS) and the activities of acid phospholipase (AP) and lipoxygenase (LOX) in drought-stressed plants were also decreased by application of Si at both stages.

MicroRNA-503 inhibits the G1/S transition by downregulating cyclin D3 and E2F3 in hepatocellular carcinoma

BackgroundIncreasing evidence indicates that deregulation of microRNAs (miRNAs) is involved in tumorigenesis. Downregulation of microRNA-503 has been observed in various types of diseases, including cancer. However, the biological function of miR-503 in hepatocellular carcinoma (HCC) is still largely unknown. In this study we aimed to elucidate the prognostic implications of miR-503 in HCC and its pathophysiologic role.MethodsQuantitative reverse transcriptase polymerase chain reaction was used to evaluate miR-503 expression in HCC tissues and cell lines. Western blotting was performed to evaluate the expression of the miR-503 target genes. In vivo and in vitro assays were performed to evaluate the function of miR-503 in HCC. Luciferase reporter assay was employed to validate the miR-503 target genes.ResultsmiR-503 was frequently downregulated in HCC cell lines and tissues. Low expression levels of miR-503 were associated with enhanced malignant potential such as portal vein tumor thrombi, histologic grade, TNM stage, AFP level and poor prognosis. Multivariate analysis indicated that miR-503 downregulation was significantly associated with worse overall survival of HCC patients. Functional studies showed miR-503 suppressed the proliferation of HCC cells by induction of G1 phase arrest through Rb-E2F signaling pathways, and thus may function as a tumor suppressor. Further investigation characterized two cell cycle-related molecules, cyclin D3 and E2F3, as the direct miR-503 targets.ConclusionOur data highlight an important role for miR-503 in cell cycle regulation and in the molecular etiology of HCC, and implicate the potential application of miR-503 in prognosis prediction and miRNA-based HCC therapy.

Antitumor activity of emodin against pancreatic cancer depends on its dual role: promotion of apoptosis and suppression of angiogenesis.

Background Emodin has been showed to induce apoptosis of pancreatic cancer cells and inhibit tumor growth in our previous studies. This study was designed to investigate whether emodin could inhibit the angiogenesis of pancreatic cancer tissues and its mechanism. Methodology/Principal Finding In accordance with our previous study, emodin inhibited pancreatic cancer cell growth, induced apoptosis, and enhanced the anti-tumor effect of gemcitabine on pancreatic caner cells in vitro and in vivo by inhibiting the activity of NF-κB. Here, for the first time, we demonstrated that emodin inhibited tumor angiogenesis in vitro and in implanted pancreatic cancer tissues, decreased the expression of angiogenesis-associated factors (NF-κB and its regulated factors VEGF, MMP-2, MMP-9, and eNOS), and reduced eNOS phosphorylation, as evidenced by both immunohistochemistry and western blot analysis of implanted tumors. In addition, we found that emodin had no effect on VEGFR expression in vivo. Conclusions/Significance Our results suggested that emodin has potential anti-tumor effect on pancreatic cancer via its dual role in the promotion of apoptosis and suppression of angiogenesis, probably through regulating the expression of NF-κB and NF-κB-regulated angiogenesis-associated factors.

Triiodothyronine attenuates hepatic ischemia/reperfusion injury in a partial hepatectomy model through inhibition of proinflammatory cytokines, transcription factors, and adhesion molecules

BACKGROUND Hepatic ischemia/reperfusion (I/R) injury during liver surgery and transplantation is the main cause of postoperative liver failure and primary graft nonfunction, with subsequent rise in mortality in these patients. Triiodothyronine (T3) is a known hepatic mitogen. In this study we questioned whether exogenous administration of T3 protects against warm hepatic I/R injury. MATERIALS AND METHODS T3 or vehicle was administered to male Sprague-Dawley rats (single dose of 0.5 mg/kg intraperitoneally) 48 h before hepatic ischemia, then the rats were subjected to 60 min of partial hepatic I/R followed by 50% hepatectomy. Serum transaminases, histopathologic changes, apoptosis, malondialdehyde, and myeloperoxidase were evaluated. The expression of proinflammatory cytokines (TNF-α, IL-1β, and IL-6), transcription factors (NF-кB and AP-1), and adhesion molecules (ICAM-1, VCAM-1) was also investigated. RESULTS Rats pretreated with T3 showed significant reduction of their postischemic hepatic injury (serum transaminases, liver necrosis, and apoptosis). Also, production of reactive oxygen species and neutrophil infiltration were markedly depressed by T3 administration. This was associated with downregulation of proinflammatory cytokines, transcription factors, and adhesion molecules. CONCLUSIONS This study illustrated that T3 protects against hepatic I/R injury, an effect that is mediated through inhibition of proinflammatory cytokines, transcription factors, and adhesion molecules. Pretreatment with T3 may represent a promising pharmacologic strategy for protection against hepatic I/R injury.

FOXC1 Contributes To Microvascular Invasion In Primary Hepatocellular Carcinoma Via Regulating Epithelial-Mesenchymal Transition

The existence of microvascular invasion (MVI) formation is one of the most important risk factors predicting poor outcome in hepatocellular carcinoma (HCC) and its mechanism remains largely unknown. Epithelial-Mesenchymal Transition (EMT) has been suggested to be involved in many steps of the invasion-metastasis cascade. To elucidate the possible contribution of EMT to MVI, we initially evaluated the expression of 8 EMT-related transcription factors (TFs) in HCC patients with or without MVI and found that FOXC1 expression was significantly higher in patients with MVI than those without MVI (P < 0.05). Knockdown of FOXC1 expression in HCC cells resulted in a partial conversion of their EMT progresses, mainly regulating the mesenchymal component. Ectopic expression of snail, twist or TGF-β1 could induce expression of FOXC1, but none of the expression of snail, twist, slug or TGF-β was consistently down-regulated in response to FOXC1 silencing, suggesting FOXC1 might operate the downstream of other EMT regulators. In addition, knockdown of FOXC1 expression led to cytoskeleton modification accompanied by decreased ability of cell proliferation, migration, and invasion. Meanwhile, some matrix metalloproteinases (MMPs) and VEGF-A were also simultaneously down-regulated. Together, our findings demonstrate that FOXC1 is one of candidate predictive markers of MVI, and that inhibition of FOXC1 expression can partially reverse EMT program, offering a potential molecular therapeutic target for reducing tumor metastasis in HCC patients.

Prediction of recurrence and survival in hepatocellular carcinoma based on two Cox models mainly determined by FoxP3+ regulatory T cells

Hepatocellular carcinoma (HCC) is an aggressive disease with poor prognosis and limited methods to predict patient survival. Immune cells infiltrating tumors is known to impact clinical outcome. Here, we investigated the prognostic significance of immune infiltration within the tumor microenvironment in 245 specimens from two independent cohorts by immunohistochemical analyses. A Cox regression model was constructed using a training cohort and validated in an independent cohort. The diagnostic accuracy was evaluated by receiver operating characteristic curve. The activation, function, and chemotaxis of intratumoral regulatory T cells (Treg) were analyzed using flow cytometry, quantitative PCR, and chemotaxis assay. We identified that the proportion of FoxP3+ cells within tumors is negatively associated with patient prognosis, whereas the proportion of interleukin (IL)-17+ cell and the number of trypase+ cells are positive predictor. The two Cox models, composed of independent predictors in multivariate analysis, provided a high diagnostic accuracy of prognosis for patients with HCC. The proportion of FoxP3+ cells showed the most significant predictive power, with the highest Cox score in the two models. Furthermore, we found Tregs from tumor with high FoxP3+ proportion were more active and powerful than the counterparts from tumor with low FoxP3+ proportion. In conclusion, two Cox models are established that have considerable clinical value in predicting tumor recurrence and survival of patients with HCC, respectively. In the both models, the proportion of Tregs among CD4+ T cells plays a central role. Cancer Prev Res; 6(6); 594–602. ©2013 AACR.

Emodin sensitizes the gemcitabine-resistant cell line Bxpc-3/Gem to gemcitabine via downregulation of NF-κB and its regulated targets.

The aim of this study was to evaluate whether emodin can overcome the chemoresistance of the gemcitabine-resistant cancer cell line (Bxpc-3/Gem) in vitro. The cell line Bxpc-3/Gem was derived from the human pancreatic cancer cell line Bxpc-3. We found that Bxpc-3/Gem cells were characterized by a series of morphological changes with a resistance index of 43.51 comparing with the parental cell line. Emodin reduced Bxpc-3/Gem cell proliferation in a dose-dependent manner. Emodin and gemcitabine combination treatments resulted in decreased cell proliferation and increased apoptosis in Bxpc-3/Gem cells. In addition, combination treatments resulted in downregulation of gene and protein expression of MDR-1 (P-gp), NF-κB, XIAP, survivin, as well as inhibition of NF-κB activity and P-gp function. These observations suggest that emodin may sensitize the pancreatic cancer gemcitabine-resistant cell line Bxpc-3/Gem to gemcitabine therapy via inhibition of survival signaling.

Perioperative dynamic alterations in peripheral regulatory T and B cells in patients with hepatocellular carcinoma

BackgroundIntratumoral and circulating regulatory T cells (Tregs) have been shown to be critical in the pathogenesis of hepatocellular carcinoma (HCC). However there is limited knowledge on the alterations of regulatory B cells (Bregs). We here investigated perioperative dynamic alterations of peripheral circulating Tregs and Bregs in HCC patients to reveal the relationship between regulatory lymphocytes and its clinical implications.Methods36 patients with HCC, 6 with chronic hepatitis B infection and 10 healthy donors were enrolled for this study. Frequencies of peripheral Tregs and Bregs were measured by flow cytometry with antibodies against CD4, CD25, CD127, CD19 and IL-10 before, and after radical surgery. Then, clinical informatics of HCC patients was achieved through Digital Evaluation Score System (DESS) for the assessment of disease severity. Finally, we analysed correlations between digitalized clinical features and kinetics of circulating regulatory lymphocytes.ResultsLevel of circulating CD4+CD25+CD127- Tregs in HCC patients was significantly lower than that in healthy donors and patients with chronic hepatitis B infection before surgery, but was increased after surgery. Preoperative level of CD19+ IL-10+ Bregs in HCC patients was also significantly lower than the other groups. However it dramatically was elevated right after surgery and remained elevated compared to controls (about 7 days after surgery, P = 0.04). Frequency of circulating Tregs was correlated with circulating leukocytes, ferritin, and clinical features suggesting tumor aggressiveness including portal vein thrombosis, hepatic vein involvement and advanced clinical stages. Frequency of circulating Bregs was associated with Hepatitis B e Antigen (HBeAg) and Hepatitis B virus (HBV) DNA copy number. In addition, DESS was significantly and positively correlated with other staging systems.ConclusionFrequencies of peripheral Tregs and Bregs in HCC patients increased after surgery. These results suggest that a postoperative combination of therapies against Tregs and Bregs may be beneficial for better outcome of HCC patients after resection.

Involvement of Endoplasmic Reticulum Stress in Capsaicin-Induced Apoptosis of Human Pancreatic Cancer Cells

Capsaicin, main pungent ingredient of hot chilli peppers, has been shown to have anticarcinogenic effect on various cancer cells through multiple mechanisms. In this study, we investigated the apoptotic effect of capsaicin on human pancreatic cancer cells in both in vitro and in vivo systems, as well as the possible mechanisms involved. In vitro, treatment of both the pancreatic cancer cells (PANC-1 and SW1990) with capsaicin resulted in cells growth inhibition, G0/G1 phase arrest, and apoptosis in a dose-dependent manner. Knockdown of growth arrest- and DNA damage-inducible gene 153 (GADD153), a marker of the endoplasmic-reticulum-stress- (ERS-) mediated apoptosis pathway, by specific siRNA attenuated capsaicin-induced apoptosis both in PANC-1 and SW1990 cells. Moreover, in vivo studies capsaicin effectively inhibited the growth and metabolism of pancreatic cancer and prolonged the survival time of pancreatic cancer xenograft tumor-induced mice. Furthermore, capsaicin increased the expression of some key ERS markers, including glucose-regulated protein 78 (GRP78), phosphoprotein kinase-like endoplasmic reticulum kinase (phosphoPERK), and phosphoeukaryotic initiation factor-2α (phospho-eIF2α), activating transcription factor 4 (ATF4) and GADD153 in tumor tissues. In conclusion, we for the first time provide important evidence to support the involvement of ERS in the induction of apoptosis in pancreatic cancer cells by capsaicin.